1. Field of the Invention
The field of the invention relates to methods of modulating at least one trait in a plant. In particular, this invention relates to the modulation of traits involved in the ethylene response in a plant, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to stress and pathogen attack. Also encompassed are transgenic plants produced by the disclosed methods.
2. Background
Ethylene (C2H4) is a gaseous plant hormone that affects myriad developmental processes and fitness responses in plants, such as germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to stress and pathogen attack (Johnson, P. R. and Ecker J. R., Annu Rev Genet. 32,227-254, 1998). Another effect of ethylene on plant growth is the so-called triple response of etiolated dicotyledoneous seedlings. This response is characterized by the inhibition of hypocotyl and root cell elongation, radial swelling of the hypocotyl, and exaggerated curvature of the apical hook. Over the past decade, genetic screens based on the triple response phenotype have identified more than a dozen genes involved in the ethylene response in plants. These genes can be divided into three distinct categories: constitutive triple response mutants (eto1, eto2 and eto3, ctr1 and ran1/ctr2); ethylene insensitive mutants (etr1, etr2, ein2, ein3, ein4, ein5, and ein6); and tissue-specific ethylene insensitive mutants (hls1, eir1, and several auxin resistant mutants).
Genetic and molecular analysis of these mutants has defined a largely linear ethylene response pathway ranging from the hormone perception at the membrane to transcriptional regulation in the nucleus. Ethylene is perceived by a family of membrane-associated receptors, including ETR1/ETR2, ERS1/ERS2 and EIN4 in Arabidopsis. Ethylene binds to its receptors via a copper cofactor, and plays a negative role, whereby ethylene binding represses activity of the receptor. In the absence of ethylene, the receptors are hypothesized to be in a functionally active form that constitutively activates a Raf-like serine/threonine kinase, CTR1, also a negative regulator of the pathway. EIN2, EIN3, EIN5, and EIN6 are positive regulators of ethylene responses, acting downstream of CTR1. EIN2 is an integral membrane protein.
Loss-of-function mutations in EIN2 cause complete ethylene insensitivity, indicating that EIN2 is a positive component essential for the ethylene responses. Ethylene signaling downstream of EIN2 is mediated by EIN3, a nuclear protein. The nuclear protein EIN3 is a transcription factor that regulates the expression of its immediate target genes such as ERF1. ERF1 belongs to a large family of AP2 domain-containing transcriptional factors that have been shown to bind to a GCC-box present in the promoters of many ethylene-inducible, defense-related genes. Thus a transcriptional cascade mediated by EIN3/EIL and ERF proteins leads to the regulation of ethylene-controlled gene expression (Wang, K. L., et al. Plant Cell 14 Suppl, S131-151, 2002). The ein3 mutants show a loss of ethylene-mediated effects including gene expression, triple response, cell growth inhibition, and reduced senescence. Conversely, overexpression of EIN3 results in constitutive ethylene responses in both wild-type and ein2 mutant backgrounds. These results demonstrate that EIN3 is both necessary and sufficient for the activation of the ethylene pathway. Biochemical studies revealed that EIN3 protein can bind to a specific sequence in the promoter of a target gene, ERF1 (Solano et al., 1998).
Although EIN3 has been shown to be an essential transcription factor mediating a diverse array of plant responses to ethylene, the mechanism of its activation by ethylene has eluded characterization. Indeed, the mechanism by which genes involved in the ethylene response of plants are regulated is poorly understood. Thus, there is a need for identification of the mechanisms whereby the ethylene response in plants is regulated. Further, there is a need for methods of modulating of the mechanisms regulating the ethylene response to control aspects of the ethylene such as senescence, fruit ripening, the stress response, germination, pathogen resistance, and leaf abscission.